Project Summary/Abstract Whether amyloid formation in stress conditions serves any beneficial purpose or is instead simply a pathologic outcome of the stress is an important open question. Elucidating the cellular triggers and physiological consequences of amyloid formation therefore represents a critical barrier to advancing our understanding of amyloid biology. The applicant?s long-term goal is to use the highly facile yeast model system to understand the stimuli that trigger amyloid formation, the mechanisms underlying this response, and its physiological significance. The central objective of this proposal is to decipher the ribosome-associated mechanisms that regulate prion formation for a variety of natural and artificial yeast prions and the impact of prion formation on gene expression and metabolism. The central hypothesis is that ribosome-associated factors, such as the ribosome-associated complex (RAC) and the nascent polypeptide-associated complex (NAC), play a central role in regulating prion formation in response to environmental stimuli and that the resulting prion phenotypes have important physiological implications. The hypothesis is based on the applicant?s published work and preliminary data. The rationale for the proposed research is that elucidating the contributions of ribosome-associated processes in prion formation is a critical step in understanding the regulation and physiological impacts of amyloid formation to facilitate pharmacological manipulation. Using the naturally occurring yeast prions [PSI+], [URE3] and [RNQ+] as models, along with artificially constructed yeast prions, this hypothesis will be tested by pursuing two related but independent specific aims: 1) Identify the roles of RAC and NAC in stress-induced protein aggregation and prion formation; and 2) Determine the impact of prion induction on gene expression and metabolism. The proposed work is innovative because it represents a substantial departure from the status quo by examining the earliest possible time-point in prionogenesis ? during synthesis of the prion-forming protein ? and by assessing the physiological consequences of prion switching. Additionally, these experiments will be enriched by the applicant?s development of an innovative new prion reporter system that enables rapid, quantitative measurements of prion formation and prion variant strength in individual cells in real time. The contribution of the proposed research is expected to be the elucidation of ribosome-associated mechanisms regulating amyloid formation and the resulting consequences on cellular physiology. This contribution will be significant because co-translational amyloid formation constitutes the earliest misfolding event against which pharmacological intervention could be targeted; thus, an understanding of the underlying triggers and regulatory mechanisms of co-translational amyloidogenesis is urgently needed to advance the field and could provide a basis for targeting mammalian homologs in pharmacological interventions of mammalian protein misfolding disorders.